Idna vaccines and methods for using the same

ABSTRACT

Described herein are iDNA vectors and vaccines and methods for using the same. The iDNA generates live attenuated vaccines in eukaryotic cells in vitro or in vivo for pathogenic RNA viruses, particularly yellow fever virus and Venezuelan equine encephalitis virus. When iDNA is injected into the vaccine recipient, RNA of live attenuated virus is generated by in vivo transcription in the recipient&#39;s tissues. This initiates production of progeny attenuated viruses in the tissues of the vaccine recipient, as well as elicitation of an effective immune response protecting against wild-type, non-attenuated virus.

GOVERNMENT INTERESTS

The inventors received material related to the subject matter of thisapplication from the U.S. government under an agreement pursuant to 15U.S.C. §3710a, accordingly the U.S. government may have certain rightsin the subject matter.

FIELD

Various embodiments described herein relate to live attenuated viralvaccines and systems and methods for making and administering suchvaccines.

BACKGROUND

In the discussion that follows, reference is made to certain structuresand/or methods. However, the following references should not beconstrued as an admission that these structures and/or methodsconstitute prior art. Applicants expressly reserve the right todemonstrate that such structures and/or methods do not qualify as priorart.

Many RNA viruses, including Yellow Fever (YF) virus and VenezuelanEquine Encephalitis (VEE) virus, are dangerous human pathogens. VEE is aCategory B and YF is a Category C Priority Pathogen as categorized byNIH/NIAID.

The VEE virus is a positive single-stranded RNA arbovirus that belongsto the Alphavirus genus of the Togaviridae family. The virus istransmitted primarily by mosquitoes, which bite an infected animal andthen bite and feed on another animal or human. VEE currently is rare inthe U.S. A major epizootic in horses occurred in Texas, but only about100 laboratory-confirmed cases in humans have been documented. However,changing climate may favor establishment of the virus in wanner areas ofthe U.S. Additionally, VEE is a potential biological weapon andbioterrorism agent.

The YF virus is also a positive single-stranded RNA arbovirus. However,unlike VEE, the YF virus belongs to the family Flaviviridae. YF diseaseoccurs mostly in Africa and South America. Human infection begins afterdeposition of viral particles through the skin by an infected mosquito.The disease is frequently severe. More moderate cases can occur as aresult of previous infection by another flavivirus. There is adifference between disease outbreaks in rural or forest areas and inurban areas (Barnett, 2007). Disease outbreaks in towns and non-nativepeople can be more serious because of higher densities of mosquitovectors and higher population densities. As of 2001, the World HealthOrganization (WHO) estimates that YF virus causes 200,000 illnesses and30,000 deaths every year in unvaccinated populations. In most cases,supportive therapy is required for YF patients. Fluid replacement,transfusion of blood derivates, and other measures are used in severecases.

Live attenuated viruses have been developed to serve as vaccines formany RNA viruses such as VEE and YF, poliomyelitis, influenza, measles,mumps, rabies, and rubella viruses. Traditional live attenuated RNAvirus vaccines comprise live attenuated RNA viruses that are injectedinto the vaccine recipient. The injected virus delivers its RNA genomeinto the cells, which results in production of viral antigens as well asprogeny attenuated viruses in the tissues of the vaccine recipient. Thisleads to the elicitation of an immune response that protects against thecounterpart non-attenuated virus.

SUMMARY

This application provides vectors comprising DNA encoding an infectiousRNA molecule and an RNA polymerase promoter, where the DNA encoding aninfectious RNA molecule is operably linked to the RNA polymerasepromoter and the infectious RNA molecule encodes a Yellow Fever (YF)virus. In certain embodiments, the YF virus is non pathogenic. Alsodescribed are vaccines for Yellow Fever (YF) comprising the vectorsdescribed above, and methods for using the vaccines to immunize againsta YF virus. Also described are homogeneous clonally purified liveattenuated virus prepared from cultured cells transfected with thevector, vaccines for YF comprising the same, and methods for using thevaccines to immunize against a YF virus.

This application also provides vectors comprising DNA encoding aninfectious RNA molecule and an RNA polymerase promoter, where the DNAencoding an infectious RNA molecule is operably linked to the RNApolymerase promoter and the infectious RNA molecule encodes a aVenezuelan Equine Encephalitis (VEE) virus. In certain embodiments, theVEE virus is non-pathogenic. Also described are vaccines for VenezuelanEquine Encephalitis (VEE) comprising the vectors described above, andmethods for using the vaccines to immunize against a VEE virus. Alsodescribed are homogeneous clonally purified live attenuated virusprepared from cultured cells transfected with the vector, vaccines forVEE comprising the same, and methods for using the vaccines to immunizeagainst a VEE virus.

This application also provides vectors comprising a DNA encoding aninfectious RNA molecule and a cytomegalovirus (CMV) RNA polymerasepromoter, where the DNA encoding an infectious RNA molecule is operablylinked to the CMV RNA polymerase promoter, the CMV RNA polymerasepromoter is located from about 12 to about 18 nucleic acid residuesupstream of the 5′ end of said DNA encoding an infectious RNA molecule,and the infectious RNA molecule encodes an attenuated RNA virus. Incertain embodiments, the attenuated RNA virus is an alphavirus or aflavivirus.

This application also provides methods for attenuating an RNA virus,comprising inserting two RNA dependent RNA promoters into the cDNAencoding the RNA virus, whereby the nucleocapsid and glycoproteins areseparately expressed from independent promoters.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of iDNA based VEE TC-83 vaccines. (A)Full-length cDNA corresponding to the TC-83 RNA genome is cloned intothe DNA containing functional DNA-dependent RNA polymerase promoter, forexample CMV promoter. Location of CMV promoter, 26S promoter, poly-A,transcription termination, and ribozyme (optional) sequences are shown.(B) Example of the modified, iDNA-based TC-83 vaccine, in which TC-83capsid and glycoprotein genes are expressed from independent promoters.Location of the promoters is shown. The transcription start site can bemodified by varying the distance between the 3′ end of the CMV promoterand the 5′ end of the TC -83 coding sequence.

FIG. 2. Administration of the TC-83 iDNA vaccine into cells in vitro orin vivo. Injection of the TC-83 DNA vaccine under control ofDNA-dependent RNA polymerase promoter (see FIG. 1) into cells in vitroor in vivo is shown. As a result of TC-83 iDNA vaccine administration,the TC-83 live attenuated virus vaccine is generated. If administered invivo, production of TC-83 vaccine in the tissues of the patient elicitsimmune response to the TC-83 vaccine.

FIG. 3. Schematic representation of iDNA-based YF17D vaccine.Full-length cDNA corresponding to the 17D ANA genome is cloned into theDNA containing functional RNA polymerase promoter, for example CMVpromoter. Location of CMV promoter, polyA, transcription termination,and ribozyme sequences are shown.

FIG. 4. Administration of the recombinant YF17D iDNA vaccine into cellsin vitro or in vivo. Injection of the 17D ‘DNA vaccine containing YF17DcDNA under control of DNA-dependent RNA polymerase promoter into cellsin vitro or in vivo is shown. As a result of 17D iDNA vaccineadministration, the 17D live attenuated virus vaccine is generated. Ifadministered in vivo to the tissues of vaccine recipient, production ofYF17D vaccine in the tissues of the patient leads to elicitation ofimmune response to the 17D vaccine.

FIG. 5. Immunofluorescense assay (IFA) of CHO cells transfected with (A)VEE TC-83 iDNA vaccine, Clone 13-2 (FIG. 6); and (B) p3-40 DNAexpressing TC-83 structural proteins (control). Focus of TC-83-positivecells is visible on panel (A), whereas panel (B) shows individualTC-83-positive cells. Cells are transfected with DNA vaccine usingFugene 6 transfection reagent or a similar gene transfer method.Transfected cells are incubated at 37° C. in 5% CO₂ incubator. Following24 hr incubation, cells are fixed with cold acetone, and IFA is doneusing rabbit antibody specific for TC-83 antigen. Then, cells areincubated using rhodamine-conjugated antibody for rabbit IgG andobserved using fluorescent microscopy.

FIG. 6. iDNA sequence fragment from pAA_TC83 plasmid (Clones #13-1;13-2) containing the TC-83 cDNA downstream from CMV promoter (SEQ ID NO:1). Locations of CMV promoter, 26 S promoter, and polyA site areindicated.

FIG. 7. iDNA sequence fragment of modified pAA_TC-83_C_GP plasmid (Clone#12) containing two TC-83 265 promoters (SEQ ID NO: 2). Locations of CMVpromoter, 26 S promoters, and polyA site are indicated.

FIG. 8. iDNA sequence fragment of pCMV_YF17D containing the YF17D cDNAdownstream from CMV promoter and hepatitis a ribozyme and BGHtranscription termination and polyadenylation cassettes downstream from3′ end of the YF17D sequence (SEQ ID NO: 3).

FIG. 9. Optimization of the distance between the 3′ end of the CMVpromoter and the 5′ end of the TC-83 cDNA by encapsidation assay usingHA- or N-vectors and DNA c-helpers.

FIG. 10. Transfection of CHO cells with TC-83 iDNA #13-1 (wild-type)results in rapid expression of TC-83 antigen in CHO cells.

FIG. 11. Transfection of CHO cells with TC-83 iDNA #12 (double 26Spromoter) results in delayed expression of TC-83 antigen in CHO cells.

FIG. 12. TC-83 viruses generated from infectious clones in vitro areavirulent in BALE/c mice. Cloned TC-83 viruses are generated bytransfection of CHO cells using electroporation with infectious TC-83vaccine cDNA clones #12 and #13-1. Viruses are inoculated in miceaccording to standard USAMRIID protocol (Dr. Michael Parker, USAMRIID,Ft, Detrick, Md.).

FIG. 13. Synthetic oligonucleotides of varying lengths (SEQ ID NOS:4-14, from top to bottom) for creating a series of “capsid iDNA”plasmids in which the distance between the promoter and the iDNA variesfrom 8 to 25 base pairs (see Example 8). The capitalized and bolded Ashows the 5′ end of the VEE sequence (in TC-38, the start codon is ATArather than ATG; see the ATA nucleotides at positions 704-706 in SEQ IDNO: 1).

DETAILED DESCRIPTION

Described herein are compositions for eliciting an immune response or aprotective immunity against Yellow Fever (YF) or Venezuelan EquineEncephalitis (VEE) viruses. In one embodiment, the compositions comprisevaccines for preventing and/or treating YF or VEE virus associateddiseases. Also described are methods of making, using and testing thesame.

Live attenuated, cell-culture derived TC-83 vaccine for VEE has beendeveloped previously. TC-83 contains attenuating mutations (Kinney etal., 1993). The current TC-83 vaccine is fully licensed for veterinaryuse in horses and was used successfully during 1970-1971 Texasepizootic. The vaccine has also been approved for use in people as anInvestigational New Drag (IND). The TC-83 vaccine provides protectionagainst many epizootic strains The TC-83 vaccine has been used as partof safety programs and was important in protecting individuals workingwith infected animals and virus preparations. To date, the vaccine hasbeen administered to ˜9,000 people. Another human IND vaccine, C-84, hasbeen prepared from formalin-inactivated TC-83 vaccine. Because of thehistory of successful use as a vaccine in people, the TC-83 vaccine isalso a promising vaccine vector, which can be used as a carrier oftherapeutic or vaccine-relevant genes (Pushko P., U.S. PatentApplication No. 2006/0198854, Vector platforms derived from thealphavinas vaccines).

Because there is no specific therapy for YF, vaccination is the onlyeffective medical countermeasure. Current YF vaccine is a live,attenuated virus preparation made from the 17D YF virus strain(Smithburn et al., 1956). The 17D live virus vaccines have beenconsidered to be safe and effective (Monath, 2001). The 17D YF vaccinevirus is the foundation for both the 17D-204 and 17DD lineages. Vaccine17D-204 is used in the U.S. and Australia, whereas vaccine 17DD is usedin Brazil. Sequencing has revealed that the 17D-204 and 17DD vaccinetypes are not identical, which reflects accumulation of geneticmutations during multiple passages of virus seeds. With safety record inhumans, the YF17D is also a promising vector for the development ofvaccines against flavivirus-related pathogens (e.g. chimeric YF17D-basedvaccines against Japanese encephalitis, dengue, and West Nile virus(Pugachev et al, 2005) as well as against pathogens outside theflavivirus genus such as malaria (Tao et al., 2005) and Lassa virus(Bredenbeek et al,, 2006).

Described herein are iDNA molecules expressing the RNA genome of liveattenuated viruses and methods for using the same. In certainembodiments, when iDNA is injected into the cultured cells in vitro, RNAof live attenuated virus is generated in the cells by in vivotranscription. This initiates production of progeny attenuated virusesin the medium from cultured cells. Such homogenous, clonally pure liveattenuated virus can be used for vaccination as improved, homogeneouslive attenuated vaccine. In other embodiments, when iDNA is injectedinto the cells of a vaccine recipient, RNA of live attenuated virus isgenerated by in vivo transcription in the tissues. This initiatesproduction of progeny attenuated viruses in the tissues of vaccinerecipient, as well as elicitation of effective immune response to liveattenuated virus. Similarly to any DNA, the iDNA can be made inbacterial cells and represents a stable molecule.

In certain embodiments, the iDNA molecules are vectors comprising DNAencoding an infectious RNA molecule, where the infectious RNA moleculein turn encodes a YF or a VEE virus. The DNA encoding an infectious RNAmolecule can be operably linked to an RNA polymerase promoter, and isgenerally modified to encode a non-pathogenic (attenuated) YF or VEEvirus.

In certain embodiments, the iDNA (infectious DNA) molecules comprise theVEE TC-83 or the VT YF 1 7D cDNA. For example, an exemplary iDNA-basedVEE vaccine comprises a DNA molecule that contains the complete cDNAcopy of the RNA genome of the TC-83 live attenuated virus. In this iDNAmolecule, the TC-83 cDNA is placed downstream from an RNA polymerasepromoter, such as the CMV promoter. When such an iDNA molecule isintroduced into cells in vitro, the TC-83 viral RNA is generated in thecells. The resulting TC-83 RNA is “infectious” and initiates productionof the TC-83 live attenuated virus vaccine in the cells. Such TC-83virus vaccine can be harvested from cultured cells and used forvaccination according to current practices. In certain embodiments, thevaccine that is generated from the TC-83 iDNA represents homogeneousprogeny virus generated from the same, well-characterized, stable DNA.Because the same, clonally purified, iDNA is used for the production ofthe vaccine lots, such vaccine will in certain embodiments have greateruniformity and lot-to-lot consistency compared to current vaccines,which can accumulate mutations during virus passages.

Alternatively, iDNA vaccine containing the TC-83 cDNA can beadministered to the vaccine recipient directly. Such iDNA administrationto the vaccine recipient initiates production of the TC-83 virus vaccinein the tissues of the patient in vivo, which provides successfulvaccination against VEE.

Similarly, iDNA-based YF vaccines can comprise a DNA molecule thatcontains the cDNA copy of the RNA genome of the YF17D live attenuatedvirus. In this iDNA molecule, the YF17D cDNA copy can be placeddownstream from an RNA polymerase promoter, for example thecytomegalovirus (CMV) promoter. When such iDNA molecule is introducedinto cells in vitro or administered directly to the tissues of vaccinerecipient in vivo, the 17D viral RNA is generated by transcription fromthe RNA polymerase promoter. The resulting YF17D RNA is infectious andinitiates production of the YF17D live attenuated vaccine. When injectedinto tissues of a vaccine recipient in vivo, such YF17D-based iDNAprovides successful vaccination of the patient against YF.

The TC-83 or YF17D cDNA can be modified to ensure sufficient attenuationand/or to introduce other characteristics, while still maintaininginfectivity and the desired therapeutic effect. In certain embodiments,the cDNA is modified by insertion, deletion, and/or substitution of oneor more of the nucleotides in the TC-83 or YF17D cDNA sequence. Forexample, the modified cDNA can have at least 50%, 60%, 70%, 80%, or 90%sequence identity with the TC-83 or YF17D sequence, such as at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

Examples of modified cDNAs include a DNA having an additional 26Spromoter in the modified TC-83 iDNA Clone 12 (see example 3, Table I).This modification slows the development of TC-83-positive foci, which isa sign of additional attenuation caused by insertion of the additional26S promoter in this construct (FIG. 1, B). Such additional attenuationcan improve the TC-83 vaccine and reduce adverse effects associated withthis vaccine. In this example, an additional 265 promoter could beinserted so that the TC-83 nucleocapsid and glycoproteins are generatedfrom independent promoters (FIG. 1B). Thus, the TC-83 26S promoter isduplicated, and the capsid and glycoproteins are generated from the two26S promoters. Other modification can be made to increase the stabilityof the iDNA in E.coli or in target cells, or to increase stability ofiDNA in E.coli cells.

Additionally, other genes or gene fragments, including genetic materialfrom other alphaviruses, or from unrelated sources such other viruses,bacteria, microorganisms, other organisms, plants, animals, or/andhumans could be inserted into the iDNA. In such cases, the modifiedTC-83 or YF17D iDNA could serve as a vector for expression ofheterologous genes in vitro or in vivo.

Described herein are specialized vectors for preparation of iDNAvaccines. However, it will be appreciated by those skilled in the artthat the iDNA described herein can be formed using any suitable vector.In general, a vector is a nucleic acid molecule (typically DNA or RNA)that serves to transfer a passenger nucleic acid sequence (i.e., DNA orRNA) into a host cell. Three common types of vectors include plasmids,phages and viruses. In an exemplary embodiment, the vector is a plasmid.The present iDNA vaccines can he comprised of DNA that is produced as aplasmid that can be introduced into animal tissue and therein isexpressed by animal cells to produce a messenger ribonucleic acid (mRNA)molecule approximately the size of the YF or the VEE genome, which istranslated to produce viral polyproteins. The viral polyproteins in turnare processed by cellular machinery to provide a full set of YF or VEEproteins that are capable of initiating replication of the above primaryRNA transcript and thus initiating the virus replication cycle in animaltissue into which the above DNA plasmid was introduced.

Suitable and exemplary plasmid vectors that have been used inconventional DNA vaccines include, but are not limited to pBR322 (ATCC#31344); pUC19 (ATCC #37254); pcDNA3.1 (Invitrogen, Carlsbad Calif.92008; Cat. NO. V385-20); pNGVL (National Gene Vector Laboratory,University of Michigan, Mich.); p414cyc (ATCC #87380), p414GALS (ATCC#87344), pBAD18 (ATCC #87393), pBLCAT5 (ATCC #77412), pBluescriptIIKS,(ATCC #87047), pBSL130 (ATCC #87145), pCM182 (ATCC #87656), pCMVtkLUC(ATCC #87633), pECV25 (ATCC #77187), pGEM-7zf (ATCC #87048), pGEX-KN(ATCC #77332), pIC20 (ATCC #87113, pUB110 (ATCC #37015), pUB18 (ATCC#37253).

The iDNA described herein is also under the control of a suitablepromoter. For eukaryotic expression, examples of suitable promotersinclude the cytomegalovirus (“CMV”) early promoter, the Rous sarcomavirus (“RSV”) LTR promoter, and the SV40 promoter.

The following describes exemplary methods for making iDNA vectors andvaccines:

The cDNA fragment corresponding to the full-length TC-83 RNA is derivedby reverse transcription and polymerase chain reaction (RT-PCR) by usingthe TC-83 viral RNA and the TC-83 sequence-specific oligonucleotideprimers. TC-83 viral RNA is extracted from the TC-83 vaccine usingphenol extraction or other methods. The TC-83-specific oligonucleotideprimers can contain additional functional elements, including, but notlimited to, restriction enzyme sites, transcription terminators,polyadenilation signals, ribozymes, etc.

Alternatively, two or more cDNA fragments encompassing the entire TC-83RNA are generated using RT-PCR. Then, such cDNA fragments are assembledwithin a single plasmid so that they comprise the full-length cDNAcorresponding to the full-length TC-83 RNA, as described in the previousparagraph.

Alternatively, the TC-83 cDNA, as described in the previous paragraphs,is generated by using chemical synthesis or a combination of chemicalsynthesis or/and PCR or/and RT-PCR.

The cDNA fragment corresponding to the full-length RNA is cloned intothe DNA containing a functional RNA polymerase promoter, for example CMVpromoter (FIG. 1A). An example of such resulting recombinant iDNAsequence is shown (FIG. 6). As a result of transcription in vitro or invivo of such iDNA, functional infectious TC-83 RNA containing one ormore attenuating mutations is generated. The distance between thepromoter and the TC-83 cDNA can be optimized to ensure the desired levelof RNA expression. In certain embodiments, the distance between theGAGCTC 3′-end of the CMV promoter (indicated at nucleotide (nt) 687 withan arrow in FIG. 6), and the 5′-end of the TC-83 cDNA (indicated atnucleotide 703 with another arrow in FIG. 6), is 15(±3) base pairs (bp).This allows efficient transcription and production of TC-83 RNA. Forcomparison, according to Invitrogen, the CMV transcription start sitewould be located at nt 697, which is 9 at from the GAGCTC 3′ end of theCMV promoter within the pcDNA3.1(−) plasmid. Similarly, the 3′ end ofthe TC-83 cDNA can be followed by ribozymes, transcription terminationsequences, poly-A, as well as other nucleotides and signals to ensureoptimal production of functionally active RNA. In a preferredembodiment, the distance between the 3′ end of TC-82 cDNA (at 12170,FIG. 6) and the poly-A site is 184 by and can vary from between 0 toabout 500 bp.

Alternatively, the TC-83 nucleocapsid and glycoproteins are expressedfrom independent promoters (FIG. 1B).

The resulting recombinant plasmid iDNA containing the TC-83 cDNA undercontrol of the RNA polymerase promoter (FIG. 1A,B) is generated andpurified from E. coli cells. Purified iDNA is then introduced intocultured eukaryotic cells, for example into Chinese hamster ovary (CHO)cells, baby hamster kidney (BHK-21), or other susceptible cells (FIG.2). DNA is administered to cells by injection, gene gun,electroporation, liposome transfection, or other gene transfer method.In the cells, the full-length infectious TC-83 RNA is generated bytranscription, which initiates the production of TC-83 live attenuatedvirus in cultured cells and release of TC-83 virus in the medium (FIG.2). The TC-83 virus is harvested from the cell cultures, formulated, andused as a VEE vaccine according to the current practice.

Alternatively, the recombinant iDNA containing the TC-83 cDNA (FIG. 1)is introduced into the vaccine recipient directly in vivo. The iDNA isadministered into vaccine recipient tissues by injection,electroporation, liposome transfection, gene gun, or other genetictransfer method. In the tissues of vaccine recipient, the full-lengthinfectious TC-83 RNA is generated by transcription, which initiatesproduction of TC-83 live attenuated virus in vivo. The TC-83 virusantigens are released from the cells in vivo in the tissues, whichinitiates induction of effective immune response to TC-83 vaccine.

Similar to the TC-83 iDNA described above, a YF17D iDNA includes theYF17D cDNA. Full-length YF17D cDNA is derived from the full-length viralRNA or assembled from two or more fragments or synthesized by chemicalmeans as described above for the TC-83, The full-length YF17D cDNA isplaced under the control of a functional RNA polymerase promoter, asshown in FIG. 4A. In certain embodiments, the distance between theGAGCTC 3′-end of the CMV promoter and the 5′-end of the TC 83 cDNA, is15(±3) by as described above for TC-83. This allows for efficienttranscription and production of YF 17D RNA. Ribozyme, transcriptiontermination and poly (A) cassettes are placed as required downstreamfrom the 3′ end of the YF17D cDNA to ensure correct transcription andpolyadenylation of functional YF17D RNA transcripts. As a result ofYF17D iDNA transcription in vitro or in vivo, functional infectiousYF17D RNA (optionally containing one or more additional attenuatingmutations) is generated. As seen in FIG. 4, transfection of BHK-21 cellswith a full-length YF17D iDNA containing YF17D cDNA under control of theCMV promoter results in transcription of infectious RNA, translation,correct post-translational processing of polyprotein, assembly andrelease of infectious YF17D particles.

As noted above, modifications can be made to the full-length YF17D aswell TC-83 cDNA constructs. Optimization of attenuation may additionallyimprove the YF17D iDNA vaccine and reduce adverse effects includingviscerotropic disease associated with YF17D vaccination (Monath, 2007).

In certain embodiments, the methods described herein compriseadministering a composition or a DNA vaccine comprising iDNA encodingfor an attenuated YF or VEE virus in an acceptable pharmaceuticalcarrier to a subject in need thereof.

The amount of iDNA present in the compositions or in the DNA vaccinesdescribed herein is preferably a therapeutically or pharmaceuticallyeffective amount. A “therapeutically effective amount” of iDNA is thatamount necessary so that the nucleotide sequence coding for the YF orVEE polypeptide performs its immunological role without causing overlynegative effects in the host to which the composition is administered.The exact amount of plasmid to be used and the composition/vaccine to beadministered will vary according to factors such as the strength of thetranscriptional and translational promoters used, the type of conditionbeing treated, the mode of administration, as well as the otheringredients in the composition. In one embodiment, the composition orthe vaccine formulation comprises from about 1 ng to about 1 mg ofplasmid

The immunogenicity of the DNA vaccines and pharmaceutical compositionscan be modified by formulating with one or more pharmaceuticallyacceptable adjuvants or imunostimulants, such as alpha-interferon,beta-interferon, gamma-interferon, granulocyte macrophage colonystimulator factor (“GM-CSF”), macrophage colony stimulator factor(“M-CSF”), interleukin 2 (“IL-2”), interleukin 12 (“IL-12”), and CpGoligonucleotides. For preparing such compositions, methods well known inthe art can be used. In certain embodiments, the iDNA is generated inE.coli cells as a plasmid DNA, containing unmethylated CpG motifs anditself constitutes an immunostimulating molecule that activates theimmune system via toll-like receptors.

Subcutaneous injection, intradermal introduction, impression through theskin, and other modes of administration such as intraperitoneal,intravenous, oral, or inhalation delivery are also suitable. Forexample, vectors containing the iDNA can be introduced into the desiredhost by methods known in the art, for example, transfection,electroporation, microinjection, microparticles, microcapsules,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lyposome fusion), use of a gene gun(particle bombardment), or a DNA vector transporter (see, e.g., Wu etal., 1992, J. Biol. Chem. 267:963.967; Wu and Wu, 1988, J. Biol. Chem.263:14621-4 4624).

As used herein, the term “treating,” “treatment” and the like are usedherein to generally mean obtaining a desired pharmacological and/orphysiological effect, and refer to a process by which the symptoms of aYF or VEE associated disease are completely eliminated or ameliorated toany clinically and/or quantitatively measurable degree. The term“preventing” refers to a process by which a YF or VEE associated diseaseis obstructed and/or delayed. The compositions and vaccines describedherein comprise iDNA (iDNA) capable of producing a live attenuatedvirus. In one embodiment, the live attenuated virus is produced in vivo.

As used herein, the term “immune response” includes a T cell response,increased serum levels of antibodies to an antigen, the presence ofneutralizing antibodies to an antigen (such as a YF or VEE polypeptide),or combinations thereof. The term “protection” or “protective immunity”includes the ability of the serum antibodies or T cell response inducedduring immunization to protect (partially or totally) against disease ordeath caused by YF or VEE viruses,

The “subject” is a vertebrate, such as a mammal. Mammals include., butare not limited to, humans, other primates, rodents, farm animals, sportanimals (horses, etc.) and pets. In certain embodiments, the subject isa human. In other embodiments, the methods find use in experimentalanimals (such as all species of monkeys), in veterinary applicationand/or in the development of animal models for disease. In certainembodiments, the vaccine is a VEE (such as TC-83) vaccine and thesubject is a horse. A “subject in need thereof” refers to any subject,patient, or individual who could benefit from the methods describedherein.

The term “therapeutically (or “pharmaceutically”) effective dose” or“therapeutically (or “pharmaceutically”) effective amount” means a doseor amount that produces the desired effect for which it is administered.The exact dose will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques.

The term “pharmaceutically acceptable” means approved by a regulatoryagency of the federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly, in humans.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the iDNA is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil, combinations thereof and the like. Suitablepharmaceutical excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, combinations thereof and the like.The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. These compositions cantake the form of solutions, suspensions, emulsion, tablets, pills,capsules, powders, sustained-release formulations, combinations thereofand the like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oralformulations can include standard carriers such as pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, combinations thereof; etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

Thus, as used herein, the term “pharmaceutically acceptable carrier”means, but is not limited to, a vehicle for containing the iDNA that canbe injected into a mammalian host without adverse effects. Suitablepharmaceutically acceptable carriers known in the art include, but arenot limited to, gold particles, sterile water, saline, glucose,dextrose, or buffered solutions, combinations thereof and the like.Carriers may include auxiliary agents including, but not limited to,diluents, stabilizers (i.e., sugars and amino acids), preservatives,wetting agents, emulsifying agents, pH buffering agents, viscosityenhancing additives, colors, combinations thereof and the like.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “vaccine” includes a plurality of such vaccinesand reference to “the dosage” includes reference to one or more dosagesand equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present disclosure isnot entitled to antedate such publication by virtue of prior disclosure.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.All publications, patents, patent applications and other referencescited herein are hereby incorporated by reference.

While the disclosure has been described in detail with reference tocertain embodiments thereof; it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scone of the disclosure. In addition, the followingexamples are illustrative of the methods described herein and should notbe considered as limiting the foregoing disclosure in any way.

EXAMPLES

Example 1. Preparation of TC-83 iDNA. Total RNA is extracted from theTC-83 vaccine using phenol extraction. The cDNA corresponding to thefrill-length TC-83 RNA is derived by reverse transcription andpolymerase chain reaction (RT-PCR) using extracted TC-83 viral RNA andthe TC-83 sequence-specific oligonucleotide primers.

The cDNA fragment corresponding to the full length TC-83 RNA is clonedinto the plasmid vector pASP5 containing a functional CMV promoter (FIG.1A, FIG. 6), which yields the TC-83 iDNA, clone 13-1 and clone 13-2. Thedistance between the 3′ end of the CMV promoter to the S′end of theTC-83 cDNA is 15 by as shown in FIG. 6. After transcription of suchTC-83 iDNA in vitro or in vivo by cellular transcription machinery,functional infectious TC-83 RNA and TC-83 virus are generated.

The TC-83 iDNA can be easily modified to optimize functionalcharacteristics, for example, the level of attenuation. For example,modified TC-83 cDNA, clone 12, is generated by duplicating the 26Spromoter and placing the second 26S promoter upstream from the TC-83glycoprotein genes (FIG. 1B, FIG. 7). in such a construct, the RNA thatis generated from the CMV promoter expresses the TC-83 capsid andglycoproteins from independent 26S promoters. In order to ensureexpression of the TC-83 proteins from such modified TC-83 RNA,appropriate changes are made, for example, ATG codon is introduced atthe 5′ terminus of the glycoprotein genes. The full-length RNA encodingcDNA of modified TC-83 (Clone 12) is introduced into the claimed plasmidvector pASP5 containing functional CMV promoter (FIG. 1B, FIG. 7), Thedistance between the 3′ end of CMV promoter to the 5′end of modifiedTC-83 cDNA is 15 bp as shown in FIG. 7.

Example 2

Preparation of YF17D iDNA. Total RNA is extracted from the YF17D vaccineusing phenol extraction or a similar method. The cDNAs corresponding tothe full-length 17D RNA is derived by reverse transcription andpolymerase chain reaction (RT-PCR) using extracted 17D viral RNA and the17D sequence-specific oligonucleotide primers. Alternatively, thefull-length YF17D cDNA is assembled from two or more plasmids.

The cDNA fragment corresponding to the full-length RNA is transferredinto the claimed plasmid vector pASP5 containing functional CMV promoter(FIG. 3), which results in YF17D iDNA (FIG. 8). The distance between the3′ end of CMV promoter to the 5′end of YF 17D cDNA is 15 bp as shown inFIG. 8 and described above for the TC-83 constructs. After transcriptionof YF17D iDNA in cells in vitro or in vivo, functional infectious YF17DRNA and YF17D virus are generated.

Example 3

Transfection of CHO cells with TC-83 1DNA vaccine. Plasmid DNAcontaining TC-83 iDNA is transfected into CHO cells using Fugene 6transfection reagent (Roche Applied Sciences). Briefly, CHO cells aregrown in 75 cm² flasks, then rinsed with phosphate buffered saline (PBS)and trypsinized. Aliquot of CHO cell suspension is transferred into6-well cell culture plates. Then, mixture of plasmid DNA and Fugene 6reagent is added according to manufacturer's instructions. As plasmidDNA, the following constructs are used

(1) VEE TC-83 modified iDNA, Clone 12 (FIG. 1B, FIG. 7);

(2) VEE TC-83 iDNA, Clone 131 (FIG. 1A, FIG. 6);

(3) VEE TC-83 iDNA, Clone 13-2 (FIG. 1A, FIG. 6);

(4) As a control, p3-10 DNA expressing TC-83 structural proteins only,is used.

As an additional control, untransfected CHO cells (5) are used. TheiDNAs (1) through (3) contain the complete TC-83 cDNA and are expectedto generate live TC-83 virus. As described above, the Clone 12 iDNA (1)contains duplicated 26S promoter to express TC-83 capsid andglycoproteins from two independent 26S promoters (FIG. 1B). In contrast,DNA (4) contain only a fragment of TC-83 corresponding to the TC-83structural genes only and is not expected to generate live TC-83 virus.

An aliquot (0.3 ml) from transfected CHO cells from 6-well plates isseeded into 8-well chamber slides. Transfected CHO cells are incubatedat 37° C. in 5% CO₂. Cell mortality is determined in 6-well plates dailyby visual microscopy. Immunofluorescense assay (IFA) is performed at 24hr posttransfection using 8-well chamber slides with antiserum thatrecognizes the TC-83 antigens, according to the method described inPushko et al. (1997). The results are shown in Table I. Cellstransfected with iDNAs (1) through (3) die within 5 daysposttransfection, whereas CHO cells with control transfections (4) and(5) remain alive. Also, foci of cells expressing TC-83 antigens aredetected by IFA at 24 hr posttransfection in the cells transfected withDNAs (2) and (3), thus indicating presence of live TC-83 virus (FIG. 5).The result indicates that introduction of iDNA-based TC-83 vaccines intocultured cells resulted in production of live TC-83 virus.

TABLE I Transfection of CHO cells with the TC-83 vaccine iDNA CHO CellFoci of Mortality after infected cells, iDNA Vaccine* DNA transfection**By IFA*** 1. VEE TC-83 modified iDNA, Yes None, only Clone 12 (FIG. 7)positive individual cells 2. VEE TC-83 iDNA, Clone Yes Yes 13-1 (FIG. 6)3. VEE TC-83 iDNA, Clone Yes Yes 13-2 (FIG. 6) 4. p3-10 DNA expressingTC-83 No None, only structural proteins only positive (control)individual cells 5. Untransfected CHO cells No No (control) *DNAtransfected into CHO cells is done in 6-well plates using Fugene 6transfection reagent (Roche Applied Sciences) **Observed on day 5 aftertransfection. ***IFA, immunofluorescence assay, by using antiserum forTC-83 structural proteins.

Example 4

Transfection of BHK-12 cells with YF17D iDNA vaccine. Briefly, plasmidDNA containing YF17D iDNA is transfected into cultured cells (CHO,BHK-21, or similar cell lines) and assayed using standard methods asdescribed in Example 3. The results are shown in Table II. Plaque assayis used to determine the titer of live YF17D virus generated in cellstransfected with plasmid iDNA. These results indicate that introductionof iDNA-based 17D vaccine into cultured cells results in synthesis ofvirus-specific RNA and in production of live YF17D virus (Table II).

TABLE II Transfection of BHK-21 cells with plasmid DNA containing YF17DiDNA BHK Cell Mortality after YF17D titer, DNA transfection by plaqueiDNA Vaccine* in vitro** assay*** 1. YF17D iDNA Yes 10⁸ pfu/ml 6.Untransfected BHK-21 cells No Not detected (control) *DNA transfectedinto BHK cells is done in 6-well plates using Fugene 6 transfectionreagent (Roche Applied Sciences) **Observed on day 5 after transfection***Assay performed on media collected from transfected cells 5 days posttransfection.

Example 5 Infection of CHO Cells with TC-83 Virus Derived fromiDNA-Transfected Cells

In order to confirm that live TC-83 virus is generated in the cellstransfected with plasmid DNA containing the complete TC-83 cDNA, themedium is harvested from transfected cells (see example 3) and used toinfect fresh CHO cells. Fresh CHO cells are infected in 8-well chamberslides with 100-fold diluted media harvested from transfected cells, andexpression of TC-83 antigens is detected by IFA at 24 hr postinfection(Table III). The results indicate that media from cells transfected withiDNA-based TC-83 vaccines contain live, infectious TC-83 virus,

TABLE III Infection of fresh CHO cells with medium collected from CHOcells transfected with iDNA containing the full-length cDNA of TC-83vaccine downstream from the CMV promoter* % cells positive for TC-83 DNAVaccine Transfected Antigen, by IFA 1. VEE TC-83 modified iDNA, Clone 12100 2. VEE TC-83 iDNA, Clone 13-1 80 3. VEE TC-83 iDNA, Clone 13-2 1004. p3-10 DNA expressing TC-83 structural 0 proteins only (control) 5.Untransfected CHO cells (control) 0 *Medium collected from transfectedcells 5 days posttransfection (Table I) is diluted 100-fold, then 100mcL are used to infect fresh CHO cells in 8-well chamber slides for 1 hrat 37° C., 5% CO₂. Then, 300 mcL of complete medium is added andincubation is continued for 24 hr. **IFA 24 hr postinfection, by usingantiserum for TC-83 structural proteins.

Example 6 Vaccination of Mice with TC-83 iDNA Vaccine

Experimental mice (BALB/c, C57BL/6, Swiss Webster outbred, or othersusceptible strain) are injected intramuscularly, subcutaneously, andintradermally with a dose of each TC-83 iDNA vaccine (Clones 12, 13-1,13-2, as indicated in Table I) ranging from 1 ng to 1 mg. The TC-83 iDNAvaccines are isolated from E.coli as plasmid DNA by using PromegaEndo-free DNA isolation method. In 30 days, animals receive a secondidentical dose of the TC-83 iDNA vaccine. Serum samples fromanesthetized animals are taken from the retroorbital sinus on days 0,30, and 60. Immune response is determined by standard immunologicalmethods including determination of serum antibody to TC-83 antigens inthe serum of vaccinated animals by ELISA. Serum antibody to TC-83antigens is detected suggesting successful vaccination with TC-83 iDNAvaccine.

Example 7 Vaccination of Mice with YF17D iDNA Vaccine

Experimental mice (BALB/c, C57BL/6, Swiss Webster outbred, or othersusceptible strain) are injected intramuscularly, subcutaneously, andintradermally with a dose of each 17D iDNA vaccine (see DNA sequence onFIG. 7) ranging from 1 ng to 1 mg. The 17D iDNA vaccine is isolated fromE.coli as plasmid DNA by using Promega Endo-free DNA isolation method.In 30 days, animals receive a second identical dose of the 17D iDNAvaccine, Serum samples from anesthetized animals are taken from theretroorbital sinus on days 0, 30, and 60. Immune response is determinedby standard immunological methods including determination of serumantibody to YF 17D antigens in the serum of vaccinated animals by ELISA.Serum antibody to YF antigens is detected suggesting successfulvaccination with YF17D iDNA vaccine,

Example 8 Optimization of Distance Between the 3′ End of CMV Promoterand the 5′ End of cDNA by Encapsidation Assay Using HA- or N-Vectors andthe DNA c-Helpers

For the successful function of the iDNA, it is important to achieveefficient transcription of the functional, full-length TC-83 RNA fromthe iDNA plasmid. Therefore, it is important to optimize transcriptionincluding the distance between the end of the promoter and the start ofthe cDNA, in order to maximize the efficacy of synthesis of functionalRNA. We construct a “small iDNA” that encodes only the capsid gene ofTC-83 virus including regulatory regions. All other TC-83 genes aredeleted from such “capsid iDNA”. Then, we insert syntheticoligonucleotides of varying lengths (see FIG. 13) between the CMVpromoter and the “capsid iDNA” using SacI site at the 3′ terminus of theCMV promoter. Thus, a series of “capsid iDNA” plasmids is made in whichthe distance between the promoter and the iDNA varies from 8 to 25 basepairs. Each capsid iDNA construct is transfected by electroporation intoCHO cells along with (1) GP-helper RNA and (2) HA-vector RNA. TheGP-helper RNA encodes the TC-83 glycoproteins, whereas HA-vector RNAencodes the HA gene of influenza. In the cotransfected CHO cells, theTC-83 capsid and GP proteins encapsidates the HA-vector intosingle-cycle particles. By titration of such particles usingimmunofluorescence assay (IFA) and HA antiserum, the titer of theparticles is detected. In this system, the level of expression of capsidfrom the “capsid iDNA” is a rate-limiting factor and affects the titerof the particles.

We find that the optimal distance between the 3′-terminus of the CMVpromoter and the 5′ end of the TC-83 capsid cDNA is 15 base pairsbetween the Sad site (the end of CMV promoter) and the start of cDNA(FIG. 9). We find that this optimal distance provided expression offunctional capsid antigen at a maximum level. However, other capsid iDNAconstructs with distances of 12 to 18 bp also provide detectable levelof expression and high titers of particles. The data are confirmed inseveral experiments using titration of HA-vector. We also confirm theseoptimization results when we use for quantitation the N-vectorexpressing nucleoprotein gene instead of HA gene.

Example 9 TC-83 Viruses Generated from Infectious Clones In Vitro areAvirulent in BALB/c Mice

TC-83 viruses are generated by transfection of CHO cells with infectiousTC-83 vaccine cDNA (iDNA), clones #12 and #13-1. Expression of TC-83antigens is shown by IFA at 24 hr post transfection in cells transfectedwith either iDNA (#12 and #13-1). Viruses are harvested from CHO cellcultures at 96 hr post transfection, after and the cytopathic effect(CPE) is apparent. The titer of each virus is determined by standardplaque assay in Vero cell monolayers. The virus generated from iDNA#13-1 has the titer of 7×107 PFU/ml, whereas the virus generated fromiDNA #12 does not show the plaques, suggesting slower formation ofplaques and possibly, higher level of attenuation. Then, 10⁵ plaqueforming units (PFU) of #13-1 virus are inoculated in BALB/c miceintramuscularly according to standard protocol. Virus generated from DNA#12 is not used for inoculation of animals, because the plaque titercannot be determined. As a control, the wild type VEE virus (Trinidadstrain) is inoculated into animals.

In the control VEE group, 10 out of 10 animals dies followinginoculation, demonstrating the virulent nature of the control virus(FIG. 12). In contrast, virus generated from iDNA (#13-1) is avirulentin mice, as mice show no signs of disease. This result confirms thatiDNA contains attenuated mutations derived from the TC-83 vaccine andthat these attenuating mutations do not revert to the wild-type virulentphenotype during virus production from iDNA.

References

Barnett E D. Yellow fever: epidemiology and prevention. Clin Infect Dis.2007 Mar. 15; 44(6):850-6. Epub 2007 Feb. 1. Review,

Bredenbeek P J, Molenkamp R, Spaan W J, Deubel V, Marian neau P, SalvatoM S, Moshkoff D, Zapata J, Tikhonov I, Patterson J, Carrion R, Ticer A,Brasky K, Lukashevich I S. A recombinant Yellow Fever 17D vaccineexpressing Lassa virus glycoproteins. Virology. 2006 Feb. 20;345(2)199-304.

Kinney R. M, Chang G J, Tsuchiya K R, Sneider J M, Roehrig J T, WoodwardT M, Trent D W. Attenuation of Venezuelan equine encephalitis virusstrain TC-83 is encoded by the 5′-noncoding region and the E2 envelopeglycoprotein, J Viral. 1993; 67(3):1269-77.

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1.-44. (canceled)
 45. A vector comprising: (a) DNA encoding aninfectious RNA molecule; and (b) a eucaryotic RNA polymerase promoter;wherein the DNA encoding an infectious RNA molecule is operably linkedto the RNA polymerase promoter.
 46. The vector of claim 45, wherein theinfectious RNA molecule encodes a non-pathogenic virus.
 47. The vectorof claim 45, wherein the RNA polymerase promoter is a cytomegalovirus(CMV) promoter.
 48. The vector of claim 47, wherein the CMV promoter islocated from about 12 to about 18 nucleic acid residues upstream of the5′ end of the DNA encoding an infectious RNA molecule.
 49. The vector ofclaim 48, wherein the CMV promoter is located 15 nucleic acid residuesupstream of the 5′ end of the DNA encoding an infectious RNA molecule.50. The vector of claim 49, wherein the vector is the pASP5 vector. 51.The vector of claim 45, wherein the vector comprises a poly-A taildownstream of the DNA encoding the infectious RNA molecule.
 52. Thevector of claim 51, wherein the poly-A tail is located from about 0 toabout 500 nucleic acid residues downstream of the 3′ end of the DNAencoding an infectious RNA molecule.
 53. A vaccine comprising atherapeutically effective amount of the vector of claim
 45. 54. Thevaccine of claim 53, further comprising a pharmaceutically acceptablecarrier.
 55. A homogeneous clonally pure live attenuated virus preparedfrom cultured cells transfected with the vector of claim
 46. 56. Avaccine for an infectious RNA virus comprising a therapeuticallyeffective amount of the homogeneous clonally pure live attenuated virusof claim
 55. 57. The vaccine of claim 56, wherein the vaccine furthercomprises a pharmaceutically acceptable carrier.
 58. A method ofpreparing clonally pure live attenuated viruses, wherein the methodcomprises transfecting the vector of claim 46 into a eukaryotic cell andisolating clonally pure infectious viruses from a culture mediumcomprising the transfected eukaryotic cell, thereby obtaining clonallypure live attenuated viruses.
 59. A method for immunizing a mammalagainst an infectious RNA virus, wherein the method comprises the stepof administering to the mammal the vaccine of claim
 54. 60. The methodof claim 59, wherein the mammal is a human or a horse.
 61. The method ofclaim 60, wherein the mammal is a human.
 62. A method of preparing avaccine for immunizing a mammal against an infectious RNA virus, whereinthe method comprises transfecting the vector of claim 1 into aeukaryotic cell and isolating clonally pure infectious viruses from aculture medium comprising the transfected eukaryotic cell, therebyobtaining a vaccine.